Multiplexing technologies have steadily gained in popularity in the fields of biology and biochemistry as the desire to perform higher-throughput and lower-cost reactions has increased. Optical multiplexing consists of attaching a known biological probe to a bead with a known optical code which allows the optical identification of each probe in the pooled sample by reading its unique optical code. In a subsequent step, the emission of a dye-labeled target is associated with the probe identity to determine the extent of the reaction in question. The two classes of materials currently used for optical multiplexing are fluorescent organic dyes and Quantum Dots but only <100 optical codes are currently available commercially (e.g. from Luminex). For the Phase I effort we hypothesized that it would be possible to obtain far more resolvable optical codes if the broad emission from organic dyes (up to 30-60 nm at FWHM) were replaced with narrow emitters such as the rare earth elements which often display emission peak widths in the 2-10 nm range. In Phase II we demonstrated that it was possible to resolve up to a billion optical codes under ideal conditions by using known amounts of rare-earth-based Parallume materials which emit up to six colors for optical multiplexing. The advantages of this system over the current flow cytometer-based systems include: spectrally discrete emission from each of the emitters, a very high level of multiplexing available through the use of variable emitter concentrations, high quantum efficiency, excellent photostability, variable particle size, and a low cost, automated parallel synthesis. Very importantly, we have also developed a completely portable, battery-powered prototype bead reader (Multiplexed Assay Reader System, or "MARS") with on superbright LEDs with an imaging system based on a very inexpensive commercial DSLR camera. In response to the specific Program Announcement, PA-08-115, for Competing Renewal applications for Complex Instrumentation under the auspices of the NHGRI, Parallel Synthesis proposes to build a complete late-stage prototype based on the Parallume platform consisting of the Parallume beads, the MARS hardware and software. Specifically, we propose to (1) develop an automated, parallel synthesis methodology for the Parallume beads to fulfill encoded set requirements, (2) create two automated, second-generation MARS prototypes and deliver them to collaborators for beta- testing resulting in feedback to create a final version of the prototype, (3) create a low-cost bead localization slide (BLS) for use in the MARS to enable high-density bead images, (4) complete a software package to control the MARS and analyze the data output from the MARS, also to be beta-tested along with the MARS, and (5) test the completely functional system by comparing our diagnostic assay for Chagas disease against a known diagnostic assay using Luminex. At the end of the three-year development period, Parallel Synthesis will have a fully functional G3 MARS with user- friendly software along with the BLS and encoded Parallume beads as consumables. This platform, offering a completely new technology for the fields of multiplex assay development and complex biological imaging and visualization, will be available for potential strategic partners, licensees, or for direct sale to end-users. This low cost system is ideally suited for low resource setting applications such as developing countries. PUBLIC HEALTH RELEVANCE: Assays used in both the developed countries and low resource settings would greatly benefit from reduced costs and increased throughput if it were possible to perform many reactions simultaneously. In order to process many pooled samples at once it is necessary to have a means of distinguishing the individual samples. This proposal describes a method by which each bead emits a unique optical signature upon excitation with a laser. The optical signature of each bead allows the determination of the bead's content and the extent of reaction during the assay. It is possible to resolve many thousands of optical codes by this method. Reading of the optical signatures by a portable detector/bead reader allows thousands of assays to be inexpensively performed and analyzed in parallel.